With the recent progress of the human genome project, the pharmacogenomics, which involves individualizing drug susceptibility at the genetic level and applying the analysis results to pharmaceutical development, has been actively studied. Diagnosis capable of individualizing drug susceptibility enables so-called personalized therapy, which allows for appropriate medication at the early stage of treatment without carrying out medication by trial and error. Particularly, molecular target drugs with fewer adverse reactions compared with conventional anticancer agents have been actively developed in the field of cancer therapy and have also started to be used in clinical practice (see for example, patent document 1). The molecular target drugs are drugs that specifically act on targeted proteins. If protein expression levels in individual patients can be profiled to determine what target protein is highly expressed, appropriate personalized therapy may be planned and practiced.
Current testing methods mainly used in clinical practice, however, are intended for the detection of single proteins and cannot simultaneously detect a large number of different proteins. Most of these testing methods employ antibodies and therefore have difficulty in conducting specific quantification tests due to cross-reaction or the like (see for example, non-patent documents 1 and 2). In addition, different antibodies must be used on a protein basis and therefore require enormous labor and cost for detecting a large number of proteins. In order to solve such problems, microarray gene expression analysis methods have been developed recently and have led to the establishment of gene expression testing methods capable of quantifying the expression of various types of RNAs at once (see for example, non-patent documents 3 to 6). Many target proteins of the molecular target drugs, however, are membrane proteins expressed on cell membranes. Since membrane protein expression levels very poorly correlate with RNA expression levels, it is not considered that RNA expression profiles obtained by microarray analysis always agree with the protein expression profiles of patients. For these reasons, personalized therapy using molecular target drugs based on the results of microarray analysis is difficult to achieve. Thus, there is a strong demand for the establishment of a testing method capable of comprehensively quantifying actually expressed proteins.
In recent years, mass spectrometry has progressed drastically and has been applied to the detection or assay of various biological materials. Mass spectrometers (MS) having various functions have been developed so far, such as electrospray ionization mass spectrometers, liquid chromatograph-mass spectrometers (LC/MS) composed of a liquid chromatograph (LC) unit connected upstream of a mass spectrometer, tandem mass spectrometers (MS/MS) composed of two mass spectrometers connected in series, and liquid chromatograph-tandem mass spectrometers (LC/MS/MS) composed of a liquid chromatograph unit connected upstream of a tandem mass spectrometer. These apparatuses are widely used in the assay or quantification of biological materials (see for example, patent documents 2 to 4).
Mass spectrometry using stable isotope labels has been developed recently and used in the detection or assay of biological materials. This method involves quantifying proteins in a sample by mass spectrometry using stable isotope-labeled proteins. According to reports, this method has been used to quantify a diagnostic marker C-reactive protein (CRP) in the serum of rheumatism patients or β-amyloid in mammalian tissue samples and body fluids. In patent documents 5 to 7, the present inventors have modified such mass spectrometry and established a method for collectively determining the absolute expression levels of membrane proteins and metabolic enzymes using LC/MS/MS. This method employs, as internal standards, stable isotope-labeled target peptide fragments consisting of the same amino acid sequence as that of target peptide fragments contained in a target protein. As a result, the absolute amount of the target peptide fragments in a biological sample digested with trypsin can be quantified. This means that the expression level of the target protein can be determined. Unfortunately, the synthesis of stable isotope-labeled peptide fragments costs around 100,000 yen per type. Thus, cost reduction has been a challenge to the method.